Tumor necrosis factor (TNF-alpha) and lymphotoxin (TNF-beta) are two closely related cytokines with a broad spectrum of biological activities. TNF-alpha and TNF-beta genes are tandemly arranged, similarly organized and separated only by 1 kb of DNA, yet they are differentially expressed in a tissue-specific manner. Control of this differential expression occurs both at the transcriptional and post- transcriptional levels. In our study we investigate how various nuclear proteins, including both transcription factors and chromatin components, can determine differential transcription of the two closely linked genes. We are using DNA transfection approach to define cis acting regulatory sequences. Our transfection data suggest the existence of a strong silencer located 1.5 kb upstream of the human TNF-beta gene promoter. Functional assay of various deletion constructs also suggests that specific organization of the TNF locus in chromatin may contribute to transcriptional control. In our study we are placing specific emphasis on NF-kB/rel family of transcription factors and we are in the process of characterizing multiple complexes formed by these proteins. TNF-alpha and TNF-beta have very similar biological effects which are modulated through common receptors. Soluble forms of the receptors are known to serve as potent inhibitors of both cytokines in vivo. Previously we observed that the cytotoxicity in the supernatants of a human B cell line corresponds to TNF-beta (lymphotoxin). Even though TNF-alpha is also expressed. We now have data indicating that the activity of TNF-alpha is completely blocked by inhibitor with specificity clearly distinguishing TNF-alpha from TNF-beta, this inhibitor is most likely the soluble form of p75 TNF receptor.